Topical compositions comprising Pichia anomala and n-acetyl glucosamine

ABSTRACT

The present invention provides a topical composition comprising an extract of  Pichia anomala  and n-acetyl glucosamine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application62/724,820 filed on Aug. 30, 2018, the complete disclosure of which ishereby incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The present invention provides a method of treating skin by topicallyapplying to skin a combination of an extract of Pichia anomala andn-acetyl glucosamine. Additionally, a topical composition comprising acombination of an extract of Pichia anomala and n-acetyl glucosamine isprovided.

BACKGROUND OF THE INVENTION

Hyaluronic acid is found in skin at the periphery of collagen andelastin fibers and where these fibers intersect. Hyaluronic acid islocalized not only in the dermis but also in the epidermal intercellularspaces, especially the middle spinous layer, but not in the stratumcorneum (SC) or stratum granulosum. In aged skin, the level ofhyaluronic acid decreases and it disassociates from collagen andelastin. Skin containing reduced levels of hyaluronic acid alsodemonstrates reduced water binding, which may be involved in the changesnoted in aged skin, including wrinkling, altered elasticity, reducedturgidity and diminished capacity to support the microvasculature of theskin. As one of the primary glycosaminoglycans (GAGs), hyaluronic acidcan bind 1000 times its weight in water, and may help the skin retainand maintain water. It is found in all connective tissue and is producedmainly by fibroblasts and keratinocytes in the skin.

Different methods have been proposed for combating wrinkles and finelines, including injection of hyaluronic acid. Injection of exogenoushyaluronic acid is used as a temporary dermal filling agent in softtissue augmentation procedures. However, injected hyaluronic acid has alimited lifetime. On the other hand, penetration of exogenous hyaluronicacid into the skin has proved difficult to accomplish by topicalapplication.

Pichia is a genus of yeasts in the family Saccharomycetaceae. More than100 species of this genus are known. The most well-known species includePichia anomala, Pichia guilliermondii, Pichia norvegensis, and Pichiaohmeri.

Pichia anomala (formerly named Hansenula anomala) can be found in rawmilk and cheese. The extracts of yeasts of the genus Pichia are rich inmannans, polysaccharides composed of mannose monomers. Pichia anomalaand mannans are known to be used in the treatment of aging skin. See,for example, FR 2938768, FR 2906719, FR 2897266 and FR 2976490.

PRO-LIPISKIN® is a commercially available cosmetic ingredient containingextract of Pichia anomala. It is produced by a Pichia strain isolatedfrom sugar cane. It is available from Silab-France.

US 2017/0172913A1 relates to topical compositions comprisingcombinations of Pichia anomala extract and chicory root extract thatprovide increased production of hyaluronic acid, along with methods oftreating signs of skin aging and improving skin barrier protection andskin moisturization.

N-acetyl glucosamine is a sugar amine and building block for GAGs. It isused in a number of cosmetic skin care products, such as NEOSTRATA SkinActive Retinol+NAG Complex commercially available from NeoStrata CompanyInc.

U.S. Pat. No. RE41339 discloses a variety of n-acetyl aldosamines,n-acetylamino acids, and other n-acetyl compounds, including n-acetylglucosamine, for topical use on skin, nails, and hair. The compoundsprovide multiple anti-aging benefits, such as alleviating thinning skin,fragile skin, fine lines, wrinkles, and so forth.

Pichia anomala extract and n-acetyl glucosamine work through differentbiological mechanisms and result in improvement of different clinicalbenefits. N-acetyl glucosamine is known as one of the substrate requiredfor HA production. Pichia anomala extract works through a hydrationpathway by increasing the activity of Hyaluronic Acid Synthase 2 enzymethat translates to the clinical benefits including hydration, improvedskin barrier, firming, etc.

Although the art provides topical uses for extracts of Pichia anomalaand n-acetyl glucosamine separately, applicants have now discovered thattopical application of a combination of these two ingredients atrelatively low amounts still unexpectedly provides excellent activityfor producing hyaluronic acid. This provides significant benefits forskin, including improving, reducing, inhibiting, or delaying theappearance of at least one sign of aging in skin, and enhancing skinbarrier protection and skin moisturization, using cost effective amountsof actives, especially the extract of Pichia anomala. Accordingly, newmethods of treating signs of skin aging, for example, are now available.

SUMMARY OF THE INVENTION

The present invention relates to a topical composition comprising anextract of Pichia anomala and n-acetyl glucosamine.

The invention also relates to a topical composition comprising about0.052 weight percent of an extract of Pichia anomala and about 2 weightpercent of n-acetyl glucosamine.

The invention also relates to a method of treating a sign of skin aging,comprising topically applying to skin in need of treatment for skinaging a topical composition comprising an extract of Pichia anomala andn-acetyl glucosamine.

The invention further provides a method of improving skin barrierfunction and moisturization, comprising topically applying to skin inneed of improving skin barrier function and moisturization a topicalcomposition comprising an extract of Pichia anomala and n-acetylglucosamine.

The invention also provides a method of increasing the amount ofhyaluronic acid produced by skin when contacted with an extract ofPichia anomala, comprising contacting the skin with a compositioncomprising the extract of Pichia anomala and n-acetyl glucosamine.

DETAILED DESCRIPTION

The topical composition of the present invention improves the productionof hyaluronic acid in the skin by synergistic action of extracts ofPichia anomala and n-acetyl glucosamine.

It is believed that one skilled in the art can, based upon thedescription herein, utilize the present invention to its fullest extent.The following specific embodiments are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Also, all publications, patentapplications, patents, and other references mentioned herein areincorporated by reference. Unless otherwise indicated, percentages usedto express amounts of ingredients are percentage by weight (i.e., %(W/W). Similarly, weight ratios used to express relative proportions ofingredients are also determined using percentage by weight (i.e., weightratios are calculated by dividing the percentage by weight of oneingredient by another). Unless stated otherwise, all ranges areinclusive of the endpoints, e.g., “from 4 to 9” includes the endpoints 4and 9.

As used herein, a “product” is optionally in finished packaged form. Inone embodiment, the package is a container such as a plastic, metal orglass tube or jar containing the composition. The product may furthercontain additional packaging such as a plastic or cardboard box forstoring such container. In one embodiment, the product comprises acomposition of the invention and contains instructions directing theuser to apply the composition to the skin or hair.

As used herein, “topically applying” means directly laying on orspreading on outer skin, the scalp, or hair, e.g., by use of the handsor an applicator such as a wipe, roller, or spray.

As used herein, “cosmetic” refers to a beautifying substance orpreparation which preserves, restores, bestows, simulates, or enhancesthe appearance of bodily beauty or appears to enhance the beauty oryouthfulness, specifically as it relates to the appearance of tissue orskin.

As used herein, “cosmetically acceptable” means that the ingredients theterm describes are suitable for use in contact with tissues (e.g., theskin or hair) without undue toxicity, incompatibility, instability,irritation, allergic response, or the like.

In certain embodiments, the compositions of the present invention aresuitable for treating signs of skin aging. As used herein, “signs ofskin aging” includes the presence of lines and wrinkles, loss ofelasticity, uneven skin, and blotchiness. In a particularly preferredembodiment, the sign of aging is the presence of lines and wrinklesand/or loss of elasticity.

As used herein, “treating signs of skin aging” refers to mitigating,reducing, preventing, improving, or eliminating the presence or signs ofskin aging described above.

As used herein, “wrinkle” includes fine lines, fine wrinkles, or coarsewrinkles. Examples of wrinkles include, but are not limited to, finelines around the eyes (e.g., “crow's feet”), forehead and cheekwrinkles, frown-lines, and laugh-lines around the mouth.

As used herein, “loss of elasticity” includes loss of elasticity orstructural integrity of the skin or tissue, including but not limited tosagging, lax and loose tissue. The loss of elasticity or tissuestructure integrity may be a result of a number of factors, includingbut not limited to disease, aging, hormonal changes, mechanical trauma,environmental damage, or the result of an application of products, suchas a cosmetics or pharmaceuticals, to the tissue.

As used herein, “uneven skin” means a condition of the skin associatedwith diffuse or mottled pigmentation, which may be classified ashyperpigmentation, such as post-inflammatory hyperpigmentation.

As used herein, “blotchiness” means a condition of the skin associatedwith redness or erythema.

As used herein, “improving the firmness of skin” means the enhancing ofthe firmness or elasticity of the skin, preventing the loss of firmnessor elasticity of skin, or preventing or treating sagging, lax and looseskin. The firmness or elasticity of the skin can be measured by use of acutometer. See Handbook Of Non-Invasive Methods And The Skin, eds. J.Serup, G. Jemec & G. Grove, Chapter 66.1 (2006). The loss of skinelasticity or firmness may be a result of a number of factors, includingbut not limited to aging, environmental damage, or the result of anapplication of a cosmetic to the skin.

As used herein, “improving the texture of skin” means the smoothing ofthe surface of the skin to remove either bumps or crevasses on the skinsurface.

As used herein, “improving the appearance of wrinkles in skin” meanspreventing, retarding, arresting, or reversing the process of wrinkleand fine line formation in skin.

As used herein, the term “safe and effective amount” means an amountsufficient to induce the desired effect, but low enough to avoid seriousside effects. The safe and effective amount of the compound, extract, orcomposition will vary with, e.g., the age, health and environmentalexposure of the end user, the duration and nature of the treatment, thespecific extract, ingredient, or composition employed, the particularcarrier utilized, and like factors.

As used herein, “skin in need of improving skin barrier function andmoisturization” means skin that is, but not limited to, lacking inmoisture, lacking in sebum, cracked, dry, itchy, scaly, xerodermic,dehydrated, lacks suppleness, lacks radiance, dull, or lacks lipids.

As described herein, applicants have discovered that topical applicationof a combination of low levels of an extract of Pichia anomala andn-acetyl glucosamine provides unexpectedly good skin barrier function,skin moisturization, and skin anti-aging benefits.

Applicants have also discovered that topical application of acomposition containing a combination of an extract of Pichia anomala andn-acetyl glucosamine enhances the endogenous hyaluronic acid (“HA”)level in skin above the level achieved using only the extract of Pichiaanomala, even at a higher amount. Topical use of such a composition canincrease the levels of hyaluronic acid to a direction found in youngerskin thereby providing the structural support to skin to reduce theappearance of signs of aging in skin.

Pichia anomala

The topical composition comprises one or more extracts of Pichiaanomala. In particular, such extracts may be extracts produced using oneof the various strains of Pichia anomala isolated from the fruit orother aerial parts of a plant. Any cosmetically acceptable extract ofPichia anomala may be used.

One example of a suitable extract of Pichia anomala is PRO-LIPISKIN,commercially available from Silab-France. It is produced from a strainof Pichia anomala present on sugar cane.

Another example of a suitable extract of Pichia anomala is produced froma strain of Pichia anomala present on fruit or leaves of Kiwi plant.

The extract of Pichia anomala may be provided as a solution containingdry matter (the extract) in the range of about 20 wt %, morespecifically 2 to 10 wt %, most specifically 3 to 7 wt %.

Solvents for such solutions include water, alcohols, glycols and thelike. In one embodiment, the solvent is at least about 90 wt % water, orat least about 95 wt % water.

N-Acetyl Glucosamine

The topical composition also contains n-acetyl glucosamine. N-acetylglucosamine has the structure:

Amounts

Any suitable amounts of Pichia anomala extract and n-acetyl glucosaminemay be used in the compositions of the present invention. Preferably,the compositions comprise safe and effective amounts of bothingredients. In particular, the amounts of Pichia anomala extract andn-acetyl glucosamine used are cosmetically acceptable and are selectedto achieve the desired treatment of skin for a particular condition,such as signs of aging, decreased barrier function, or decreasedmoisturization.

In certain preferred embodiments, the compositions comprise from about0.01 to about 1% by weight of Pichia anomala extract, more preferablyabout 0.025 to about 0.25% by weight of Pichia anomala extract. In oneembodiment, the composition comprises about 0.052% by weight of Pichiaanomala extract.

In certain preferred embodiments, the compositions comprise from about0.01 to about 10% by weight, more preferably about 0.5 to about 5% byweight, of n-acetyl glucosamine. In one embodiment, the compositioncomprises about 2% by weight of n-acetyl glucosamine.

In certain embodiments, the weight ratio of Pichia anomala extract ton-acetyl glucosamine in the compositions is from about 0.01 to about0.05. In one embodiment, the weight ratio of Pichia anomala extract ton-acetyl glucosamine in the compositions is about 0.026.

Topical Compositions

The compositions of the present invention are applied topically to humanskin or hair. Accordingly, the composition may further include acosmetically acceptable topical carrier that may be from about 50% toabout 99.99%, by weight, of the composition (e.g., from about 80% toabout 99%, by weight, of the composition). In a preferred embodiment ofthe invention, the cosmetically acceptable topical carrier includeswater.

The compositions may be made into a wide variety of product types thatinclude but are not limited to lotions, creams, gels, sticks, sprays,ointments, cleansing liquid washes and solid bars, shampoos and hairconditioners, hair fixers, pastes, foams, powders, mousses, shavingcreams, wipes, patches, hydrogels, film-forming products, facial masksand skin masks, films and make-up such as foundations, and mascaras.These product types may contain several types of cosmetically acceptabletopical carriers including, but not limited to solutions, suspensions,emulsions such as microemulsions and nanoemulsions, gels, solids andliposomes. The following are non-limiting examples of such carriers.Other carriers can be formulated by those of ordinary skill in the art.

The compositions useful in the present invention can be formulated assolutions. Solutions typically include an aqueous or organic solvent(e.g., from about 50% to about 99.99% or from about 90% to about 99% ofa cosmetically acceptable aqueous or organic solvent). Examples ofsuitable organic solvents include propylene glycol, polyethylene glycol,polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters,1,2,6-hexanetriol, ethanol, and mixtures thereof.

Compositions useful in the subject invention may be formulated as asolution comprising an emollient. Such compositions preferably containfrom about 2% to about 50% of an emollient(s). As used herein,“emollients” refer to materials used for the prevention or relief ofdryness, such as by preventing the transepidermal loss of water from theskin. Examples of emollients include those known in the art. Examples ofparticularly suitable emollients include vegetable oils, mineral oils,fatty esters, and the like.

A lotion can be made from such a solution. Lotions typically containfrom about 1% to about 20% (e.g., from about 5% to about 10%) of anemollient(s) and from about 50% to about 90% (e.g., from about 60% toabout 80%) of water.

Another type of product that may be formulated from a solution is acream. A cream typically contains from about 5% to about 50% (e.g., fromabout 10% to about 20%) of an emollient(s) and from about 45% to about85% (e.g., from about 50% to about 75%) of water.

The composition of the present invention may include water oralternatively be anhydrous or be an ointment that includes no water butorganic and/or silicone solvents, oils, lipids and waxes. An ointmentmay contain a simple base of animal or vegetable oils or semi-solidhydrocarbons. An ointment may contain from about 2% to about 10% of anemollient(s) plus from about 0.1% to about 2% of a thickening agent(s).

The composition may be formulated as an emulsion. If the topical carrieris an emulsion, from about 1% to about 10% (e.g., from about 2% to about5%) of the topical carrier contains an emulsifier(s). Emulsifiers may benonionic, anionic or cationic. Examples of emulsifiers are well known inthe art.

Lotions and creams can be formulated as emulsions. Typically, suchlotions contain from 0.5% to about 5% of an emulsifier(s). Such creamstypically contain from about 1% to about 20% (e.g., from about 5% toabout 10%) of an emollient(s); from about 20% to about 80% (e.g., from30% to about 70%) of water; and from about 1% to about 10% (e.g., fromabout 2% to about 5%) of an emulsifier(s).

Single emulsion skin care preparations, such as lotions and creams, ofthe oil-in-water type and water-in-oil type are well-known in thecosmetic art and are useful in the subject invention. Multiphaseemulsion compositions, such as the water-in-oil-in-water type or theoil-in-water-in-oil type, are also useful in the subject invention. Ingeneral, such single or multiphase emulsions contain water, emollients,and emulsifiers as essential ingredients.

The compositions of this invention can also be formulated as a gel(e.g., an aqueous, alcohol, alcohol/water, or oil gel using a suitablegelling agent(s)). Suitable gelling agents for aqueous and/or alcoholicgels include, but are not limited to, natural gums, acrylic acid andacrylate polymers and copolymers, and cellulose derivatives (e.g.,hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gellingagents for oils (such as mineral oil) include, but are not limited to,hydrogenated butylene/ethylene/styrene copolymer and hydrogenatedethylene/propylene/styrene copolymer. Such gels typically containbetween about 0.1% and 5%, by weight, of such gelling agents.

In one embodiment, the composition is a gel cream. The gel creamaesthetic is characterized with a watery break, semi-translucent aspectand light after-feel. As used herein, the term “gel cream” means aformulation with low levels of oil droplets suspended in aqueous gelmatrix.

The compositions of the present invention can also be formulated into asolid formulation (e.g., a wax-based stick, soap bar composition,powder, or a wipe containing powder).

The compositions may contain, in addition to the components above, awide variety of additional oil-soluble materials and/or water-solublematerials conventionally used in compositions for use on skin and hair,at their art-established levels.

Additional Cosmetically Active Agents

The compositions of the present invention may further comprise any of avariety of additional cosmetically active agents. Examples of suitableadditional active agents include: skin lightening agents, darkeningagents, additional anti-aging agents, tropoelastin promoters, collagenpromoters, anti-acne agents, shine control agents, anti-microbial agentssuch as anti-yeast agents, anti-fungal, and anti-bacterial agents,anti-inflammatory agents, anti-parasite agents, external analgesics,sunscreens, photoprotectors, antioxidants, keratolytic agents,detergents/surfactants, moisturizers, nutrients, vitamins, energyenhancers, anti-perspiration agents, astringents, deodorants, hairremovers, hair growth enhancing agents, hair growth delaying agents,firming agents, hydration boosters, efficacy boosters, anti-callousagents, agents for skin conditioning, anti-cellulite agents,odor-control agents such as odor masking or pH-changing agents, and thelike.

Examples of various suitable additional cosmetically acceptable activesinclude hydroxy acids; benzoyl peroxide; D-panthenol; UV filters such asbut not limited to avobenzone (PARSOL 1789), bisdisulizole disodium (NEOHELIOPAN AP), diethylamino hydroxybenzoyl hexyl benzoate (UVINUL APlus), ecamsule (MEXORYL SX), methyl anthranilate, 4-aminobenzoic acid(PABA), cinoxate, ethylhexyl triazone (UVINULT 150), homosalate,4-methylbenzylidene camphor (PARSOL 5000), octyl methoxycinnamate(Octinoxate), octyl salicylate (Octisalate), padimate O (ESCALOL 507),phenylbenzimidazole sulfonic acid (ENSULIZOLE), polysilicone-15 (PARSOLSLX), trolamine salicylate, Bemotrizinol (TINOSORB S), benzophenones1-12, dioxybenzone, drometrizole trisiloxane (MEXORYL XL), iscotrizinol(UVASORB HEB), octocrylene, oxybenzone (EUSOLEX 4360), sulisobenzone,bisoctrizole (TINOSORB M), titanium dioxide, zinc oxide; carotenoids;free radical scavengers; spin traps; retinoids and retinoid precursorssuch as retinol, retinoic acid and retinyl palmitate; ceramides;polyunsaturated fatty acids; essential fatty acids; enzymes; enzymeinhibitors; minerals; hormones such as estrogens; steroids such ashydrocortisone; 2-dimethylaminoethanol; copper salts such as copperchloride; peptides containing copper, coenzyme Q10; amino acids such aproline; vitamins; lactobionic acid; acetyl-coenzyme A; niacin;riboflavin; thiamin; ribose; electron transporters such as NADH andFADH2; and other botanical extracts such as oat, aloe vera, Feverfew,Soy, Shiitake mushroom extracts, and derivatives and mixtures thereof.

In certain preferred embodiments, the compositions comprise acombination of Pichia anomala extract and n-acetyl glucosamine and atleast one additional skin moisturizing active agent.

In certain preferred embodiments, the skin care compositions comprisethe combination of Pichia anomala extract, n-acetyl glucosamine, and atleast one additional agent for improving the appearance of at least onesign of aging in skin. Examples of suitable additional agents improvingthe appearance of at least one sign of aging in skin include, but arenot limited to, tropoelastin promoters, collagen promoters, retinoids,hyaluronic acid including cross-linked hyaluronic acid,dimethylaminoethanol,N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine, alpha hydroxy acids,polyhydroxyacids, and combinations of two or more thereof.

“Tropoelastin promoters,” as used herein, refers to a class of compoundsthat possess the biological activity of enhancing the production oftropoelastin. Tropoelastin promoters, according to the presentinvention, include all natural or synthetic compounds that are capableof enhancing the production of tropoelastin in the human body.

Examples of suitable tropoelastin promoters include, but are not limitedto, blackberry extracts, cotinus extracts, feverfew extracts, andbimetal complexes having copper and/or zinc constituents. The bimetalcomplex having copper and/or zinc constituents may be, for example,copper-zinc citrate, copper-zinc oxalate, copper-zinc tartarate,copper-zinc malate, copper-zinc succinate, copper-zinc malonate,copper-zinc maleate, copper-zinc aspartate, copper-zinc glutamate,copper-zinc glutarate, copper-zinc fumarate, copper-zinc glucarate,copper-zinc polyacrylic acid, copper-zinc adipate, copper-zinc pimelate,copper-zinc suberate, copper-zinc azealate, copper-zinc sebacate,copper-zinc dodecanoate, or combinations thereof. In a preferredembodiment, the tropoelastin promoter is selected from blackberryextracts, cotinus extracts, feverfew extracts, and combinations thereof.In a particularly preferred embodiment, the tropoelastin promoter isselected from blackberry extracts, feverfew extracts, and combinationsthereof.

By “blackberry extract,” it is meant a blend of compounds isolated fromthe plant of the genus Rubus, and preferably Rubus fruticosus. In oneembodiment, the compounds are isolated from the flowers of the plant. Ina further embodiment, the compounds are isolated from dried flowers ofthe plant. Such compounds may be isolated from one or more part of theplant (e.g., the whole plant, flower, seed, root, rhizome, stem, fruitand/or leaf of the plant). In a preferred embodiment, the blackberryextract is a blackberry leaf extract. One particularly suitableblackberry extract is produced by extracting the leaves of Rubusfruticosus with a mixture of water and ethanol compounded to an activityof about 5% to about 10%, with a maltodextrin matrix, commerciallyavailable from Symrise Inc. of Teterboro, N.J., and is sold under thename SYMMATRIX.

Compositions of the present invention may include a cosmeticallyeffective amount of one or more tropoelastin promoters such as thosedescribed above. The compositions preferably include, on an activebasis, from about 0.1% to about 10% of the tropoelastin promoters, morepreferably from about 0.5% to about 5% of tropoelastin promoters, andmost preferably from about 0.5% to about 2% of the tropoelastinpromoters.

“Collagen promoter,” as used herein, refers to compounds that possessthe biological activity of enhancing the production of collagen.“Non-retinoid collagen promoters” according to the present inventioninclude all natural or synthetic compounds that are not retinoids, orderived from retinoids, and are capable of enhancing the production ofcollagen in the human body.

Examples of suitable collagen promoters include, but are not limited tothe following: Retinoids including retinol, retinaldehyde, and retinoicacid, extracts of feverfew (Tanacetum parthenium), extracts of Centellaasiatica, and extracts of Siegesbeckia orientalis; extracts of soy;collagen-promoting peptides; ursolic acid; and asiaticoside. Centellaasiatica, also known as Violette marronne on Reunion Island, Gotu Kolaor Indian pennywort in India, Centella repanda in North America, andTalapetraka in Madagascar, is a polymorphous herb and belongs to thefamily of Umbelliferae (Apiaceae), particularly to the Hydrocotylesubfamily. It grows wild throughout the tropics and prefers moist andshady regions at an altitude of about 600 to 1200 meters above sealevel. Centella asiatica has three varieties: Typica, Abyssinica, andFloridana. The herb is known and used for its healing, sedative,analgesic, antidepressant, antiviral and antimicrobial properties. Thebiological activity of the herb appears to be due to the presence oftriterpene molecules in the herb. A suitable extract of Centellaasiatica is available as TECA from Bayer Consumer HealthCare of Basel,Switzerland.

By “extracts of Siegesbeckia orientalis,” is meant any of variousextracts of the plant Siegesbeckia orientalis, including Darutosideavailable from Sederma (Croda International Group of Edison, N.J.).

Suitable collagen-promoting peptides include the following matrikinepeptides, (i.e., a peptide derived from the degradation of extracellularmatrix proteins—collagen, elastin, or proteoglycan) including palmitoylpentapeptides, such as MATRIXYL from Sederma (Croda International Groupof Edison, N.J.); GHK copper peptide available as PROCYTE fromPhotomedex of Montgomeryville, Pa.; Palmitoyl GHK peptide available asBiopoeptide CL from Sederma (Croda International Group of Edison, N.J.);Biomimetic tetrapeptides, such as those available as Chronoline TriPeptide from Unipex of Québec, Canada; and Palmitoyl tri-peptide,available as Syn-Coll from DSM of Basel, Switzerland.

Ursolic acid is also known as pentacyclic triterpene acid, Prunol,Malol, Urson, beta-ursolic acid and 3-Beta-Hydroxy-Urs-12-En-28-OicAcid. It is commercially available for example from Sigma-Aldrich of St.Louis, Mo.

Asiaticoside, also known chemically as:[6-[[3,4-dihydroxy-6-(hydroxynnethyl)-5-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl]10,11-dihydroxy-9-(hydroxynnethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylate)is commercially available for example from Bayer Sante FamilialeDivision Serdex, 69, Boulevard Victor Hugo 93400 SAINT-OUEN France.

Compositions of the present invention may include a cosmeticallyeffective amount of one or more collagen promoters. The compositionspreferably include, on an active basis, from about 0.1% to about 10% ofthe collagen promoters, more preferably from about 0.5% to about 5% ofcollagen promoters, and most preferably from about 0.5% to about 2% ofthe collagen promoters.

The compositions of the present invention may comprise additionally atleast one skin lightening active agent. Examples of suitable skinlightening active agents include, but are not limited to, tyrosinaseinhibitors, melanin-degradation agents, melanosome transfer inhibitingagents including PAR-2 antagonists, exfoliants, sunscreens, retinoids,antioxidants, Tranexamic acid, tranexamic acid cetyl esterhydrochloride, skin bleaching agents, linoleic acid, adenosinemonophosphate disodium salt, Chamomilla extract, allantoin, opacifiers,talcs and silicas, zinc salts, and the like, and other agents asdescribed in Solano et al. Pigment Cell Res. 19 (550-571) and Ando etal. Int J Mol Sci 11 (2566-2575).

Examples of suitable tyrosinase inhibitors include but, are not limitedto, Vitamin C and its derivatives, Vitamin E and its derivatives, KojicAcid, Arbutin, resorcinols, hydroquinone, Flavones e.g. Licoriceflavanoids, Licorice root extract, Mulberry root extract, DioscoreaCoposita root extract, Saxifraga extract and the like, Ellagic acid,Salicylates and derivatives, Glucosamine and derivatives, Fullerene,Hinokitiol, Dioic acid, 5,5′-dipropyl-biphenyl-2,2′-diol (Magnolignan),4-(4-hydroxyphenyl)-2-butanol (4-HPB), combinations of two or morethereof, and the like. Examples of vitamin C derivatives include, butare not limited to, ascorbic acid and salts, Ascorbic Acid-2-Glucoside,sodium ascorbyl phosphate, magnesium ascorbyl phosphate, and naturalextract enriched in vitamin C. Examples of vitamin E derivativesinclude, but are not limited to, alpha-tocopherol, beta, tocopherol,gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol,gamma-tocotrienol, delta-tocotrienol and mixtures thereof, tocopherolacetate, tocopherol phosphate and natural extracts enriched in vitamin Ederivatives. Examples of resorcinol derivatives include, but are notlimited to, resorcinol, 4-substituted resorcinols like4-alkylresorcinols such as 4-butyresorcinol (rucinol), 4-hexylresorcinol(Synovea HR, Sytheon), phenylethyl resorcinol (Symwhite, Symrise),1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)-Propane(nivitol, Unigen) and the like and natural extracts enriched inresorcinols. Examples of salicylates include, but are not limited to,4-methoxy potassium salicylate, salicylic acid, acetylsalicylic acid,4-methoxysalicylic acid and their salts. In certain preferredembodiments, the tyrosinase inhibitors include a 4-substitutedresorcinol, a vitamin C derivative, or a vitamin E derivative. In morepreferred embodiments, the tyrosinase inhibitor comprises Phenylethylresorcinol, 4-hexyl resorcinol, or ascorbyl-2-glucoside.

Examples of suitable melanin-degradation agents include, but are notlimited to, peroxides and enzymes such as peroxidases and ligninases. Incertain preferred embodiments, the melanin-inhibiting agents include aperoxide or a ligninase.

Examples of suitable melanosome transfer inhibiting agents includingPAR-2 antagonists such as soy trypsin inhibitor or Bowman-BirkInhibitor, Vitamin B3 and derivatives such as Niacinamide, Essentialsoy, Whole Soy, Soy extract. In certain preferred embodiments, themelanosome transfer inhibiting agents includes a soy extract orniacinamide.

Examples of exfoliants include, but are not limited to, alpha-hydroxyacids such as lactic acid, glycolic acid, malic acid, tartaric acid,citric acid, or any combination of any of the foregoing, beta-hydroxyacids such as salicylic acid, polyhydroxy acids such as lactobionic acidand gluconic acid, and mechanical exfoliation such as microdermabrasion.In certain preferred embodiments, the exfoliants include glycolic acidor salicylic acid.

Examples of retinoids include, but are not limited to, retinol (VitaminA alcohol), retinal (Vitamin A aldehyde), retinyl acetate, retinylpropionate, retinyl linoleate, retinoic acid, retinyl palmitate,isotretinoin, tazarotene, bexarotene, Adapalene, combinations of two ormore thereof and the like. In certain preferred embodiments, theretinoid is selected from the group consisting of retinol, retinal,retinyl acetate, retinyl propionate, retinyl linoleate, and combinationsof two or more thereof. In certain more preferred embodiments, theretinoid is retinol.

Examples of antioxidants include, but are not limited to, water-solubleantioxidants such as sulfhydryl compounds and their derivatives (e.g.,sodium metabisulfite and N-acetyl-cysteine, glutathione), lipoic acidand dihydrolipoic acid, stilbenoids such as resveratrol and derivatives,lactoferrin, iron and copper chelators and ascorbic acid and ascorbicacid derivatives (e.g., ascobyl-2-glucoside, ascorbyl palmitate andascorbyl polypeptide). Oil-soluble antioxidants suitable for use in thecompositions of this invention include, but are not limited to,butylated hydroxytoluene, retinoids (e.g., retinol and retinylpalmitate), tocopherols (e.g., tocopherol acetate), tocotrienols, andubiquinones. Natural extracts containing antioxidants suitable for usein the compositions of this invention, include, but not limited to,extracts containing flavonoids and isoflavonoids and their derivatives(e.g., genistein and diadzein), extracts containing resveratrol and thelike. Examples of such natural extracts include grape seed, green tea,black tea, white tea, pine bark, feverfew, parthenolide-free feverfew,oat extracts, blackberry extract, cotinus extract, soy extract, pomeloextract, wheat germ extract, Hesperedin, Grape extract, Portulacaextract, Licochalcone, chalcone, 2,2′-dihydroxy chalcone, Primulaextract, propolis, and the like.

The additional cosmetically active agent may be present in a compositionin any suitable amount, for example, in an amount of from about 0.0001%to about 20% by weight of the composition, e.g., about 0.001% to about10% such as about 0.01% to about 5%. In certain preferred embodiments,in an amount of 0.1% to 5% and in other preferred embodiments from 1% to2%.

Compositions of the present invention may include a cosmeticallyeffective amount of one or more anti-inflammatory compounds.

Examples of suitable anti-inflammatory agents include substitutedresorcinols, (E)-3-(4-methylphenylsulfonyl)-2-propenenitrile (such as“Bay 11-7082,” commercially available from Sigma-Aldrich of St. Louis,Mo.), tetrahydrocurcuminoids (such as Tetrahydrocurcuminoid CG,available from Sabinsa Corporation of Piscataway, N.J.), extracts andmaterials derived from the following: Phellodendron amurense CortexExtract (PCE), Non-Denatured Soy (Glycine max), Feverfew (Tanacetumparthenium), Ginger (Zingiber officinale), Ginko (Ginkgo biloba),Madecassoside (Centella asiatica extract ingredient), Cotinus (Cotinuscoggygria), Butterbur Extract (Petasites hybridus), Goji Berry (Lyciumbarbarum), Milk Thistle Extract (Silybum marianum), Honeysuckle(Lonicera japonica), Basalm of Peru (Myroxylon pereirae), Sage (Salviaofficinalis), Cranberry Extract (Vaccinium oxycoccos), Amaranth Oil(Amaranthus cruentus), Pomegranate (Punica granatum), Yerbe Mate (Ilexparaguariensis Leaf Extract), White Lily Flower Extract (Liliumcandidum), Olive Leaf Extract (Olea europaea), Phloretin (appleextract), Oat Flour (Aveena sativa), Lifenol (Hops: Humulus lupulus)Extract, Bugrane P (Ononis spinosa), Licochalcone (Licorice: Glycyrrhizainflate extract ingredient), Symrelief (Bisabolol and Ginger extract),combinations of two or more thereof, and the like.

In one embodiment, the anti-inflammatory agent is a resorcinol.Particularly suitable substituted resorcinols include 4-hexyl resorcinoland 4-octylresorcinol, particularly 4-hexyl resorcinol. 4-Hexylresorcinol is commercially available as SYNOVEA HR from Sytheon ofLincoln Park, N.J. 4-Octylresorcinol is commercially available from CityChemical LLC of West Haven, Conn.

By “extracts of feverfew,” it is meant extracts of the plant “Tanacetumparthenium,” such as may be produced according to the details set forthe in US Patent Application Publication No. 2007/0196523, entitled“PARTHENOLIDE FREE BIOACTIVE INGREDIENTS FROM FEVERFEW (TANACETUMPARTHENIUM) AND PROCESSES FOR THEIR PRODUCTION.” One particularlysuitable feverfew extract is commercially available as about 20% activefeverfew, from Integrated Botanical Technologies of Ossining, N.Y.

In the skin care composition of the invention, the ratio of the amountsof the combined Pichia anomala extract and n-acetyl glucosamine to theanti-inflammatory compound may be varied. For example, the extract andthe anti-inflammatory compound may be present in a weight ratio (whichis determined by dividing the amount by weight of the dry extract by theamount by weight of the anti-inflammatory compound) of about 0.001 toabout 100, preferably about 0.01 to about 10, more preferably about 0.25to about 2.

A variety of other materials may also be present in the compositions ofthe present invention. In certain preferred embodiments, the compositioncomprises one or more topical ingredients selected from the groupconsisting of: surfactants, chelating agents, emollients, humectants,conditioners, preservatives, opacifiers, fragrances and the like.

What is meant by an emollient is a compound that helps to maintain thesoft, smooth, and pliable appearance of the skin (e.g., by remaining onthe skin surface or in the stratum corneum to act as a lubricant).Examples of suitable emollients include those found in Chapter 35, pages399-415 (Skin Feel Agents, by G Zocchi) in Handbook of Cosmetic Scienceand Technology (edited by A. Barel, M. Paye and H. Maibach, Published in2001 by Marcel Dekker, Inc New York, N.Y.), and include, but are notlimited to, petrolatum, hexyldecyl stearate and plant, nut, andvegetable oils such as macadamia nut oil, rice bran oil, grape seed oil,palm oil, prim rose oil, hydrogenates peanut oil, and avocado oil.

What is meant by a humectant is a compound intended to increase thewater content of the top layers of skin (e.g., hygroscopic compounds).Examples of suitable humectants include those found Chapter 35, pages399-415 (Skin Feel Agents, by G Zocchi) in Handbook of Cosmetic Scienceand Technology (edited by A. Barel, M. Paye and H. Maibach, Published in2001 by Marcel Dekker, Inc New York, N.Y.) and include, but are notlimited to, glycerin, sorbitol or trehalose (e.g., α,α-trehalose, β,β-trehalose, α, β-trehalose) or a salt or ester thereof (e.g., trehalose6-phosphate).

In one embodiment, the composition contains glycerin. For example, thecomposition contains at least about 4 wt % glycerin. The composition maycontain at least about 10 wt % glycerin.

In another embodiment, the composition has a pH of about 6.5 or less.For example, the composition may have a pH of about 5.5 to about 6.

In a particular embodiment, the composition contains about 0.052 weightpercent of an extract of Pichia anomala and about 2 weight percent ofn-acetyl glucosamine.

The composition may further contain about 1 to about 2 weight percent ofa mixture of cetearyl olivate and sorbitan olivate, about 4 to about 5weight percent glycerin, and at least about 65 weight percent water, andhave a pH of less than about 6.

What is meant by a surfactant is a surface-active agent intended tocleanse or emulsify. Examples of suitable surfactants include thosefound in Chapter 37, pages 431-450 (Classification of surfactants, by L.Oldenhove de Guertechin) in Handbook of Cosmetic Science and Technology(edited by A. Barel, M. Paye and H. Maibach, Published in 2001 by MarcelDekker, Inc New York, N.Y.) and include, but are not limited to anionicsurfactants such as sulfates, cationic surfactants such as betaines,amphoteric surfactants such as sodium coco glycinate, noionicsurfactants such as alkyl polyglucosides.

Examples of suitable chelating agents include those which are capable ofprotecting and preserving the compositions of this invention.Preferably, the chelating agent is ethylenediamine tetracetic acid(“EDTA”), and more preferably is tetrasodium EDTA, availablecommercially from Dow Chemical Company of Midland, Mich. under the tradename VERSENE 100XL.

Suitable preservatives include, for example, parabens, quaternaryammonium species, phenoxyethanol, benzoates, DMDM hydantoin, organicacids and are present in the composition in an amount, based upon thetotal weight of the composition, from about 0 to about 1 percent or fromabout 0.05 percent to about 0.5 percent.

Any of a variety of conditioners which impart additional attributes,such as gloss to the hair, are suitable for use in this invention.Examples include, but are not limited to, volatile silicone conditioningagent having an atmospheric pressure boiling point less than about 220°C. Examples of suitable volatile silicones nonexclusively includepolydimethylsiloxane, polydimethylcyclosiloxane, hexamethyldisiloxane,cyclomethicone fluids such as polydimethylcyclosiloxane availablecommercially from Dow Corning Corporation of Midland, Mich. under thetradename, “DC-345” and mixtures thereof, and preferably includecyclomethicone fluids. Other suitable conditioners include cationicpolymers, including polyquarterniums, cationic guar, and the like.

Any of a variety of commercially available pearlescent or opacifyingagents are suitable for use in the composition. Examples of suitablepearlescent or opacifying agents include, but are not limited to, monoor diesters of (a) fatty acids having from about 16 to about 22 carbonatoms and (b) either ethylene or propylene glycol; mono or diesters of(a) fatty acids having from about 16 to about 22 carbon atoms (b) apolyalkylene glycol of the formula: HO—(JO)_(a)—H, wherein J is analkylene group having from about 2 to about 3 carbon atoms; and a is 2or 3; fatty alcohols containing from about 16 to about 22 carbon atoms;fatty esters of the formula: KCOOCH₂L, wherein K and L independentlycontain from about 15 to about 21 carbon atoms; inorganic solidsinsoluble in the shampoo composition, and mixtures thereof.

Any fragrance compositions suitable for use on skin may be used inaccord with the present invention.

In certain preferred embodiments, the present invention is in the formof a substrate comprising a composition of the present invention. Anysuitable substrate may be used. Examples of suitable substrates andsubstrate materials are disclosed, for example, in U.S. PublishedApplication Nos. 2005/0226834 and 2009/0241242 which are incorporatedherein by reference in their entirety.

In certain preferred embodiments, the substrate is a wipe, glove, or afacial mask. Preferably, such embodiments comprise a water-insolublesubstrate as such is defined in the cited references above. For certainembodiments, the water-insoluble substrate may have a size and shapesuch that it covers the face of a human user to facilitate placing thewater-insoluble substrate about the face of the user as a masksubstrate. For example, the water-insoluble mask substrate may haveopenings for a mouth, nose, and/or eyes of the user. Alternatively, thewater-insoluble substrate may have no such openings. Such aconfiguration without openings may be useful for embodiments of theinvention in which the water-insoluble substrate is intended to bedraped over a non-facial expanse of skin or if the water-insolublesubstrate is intended to be used as wipe. The water-insoluble substratemay have various shapes, such as an angular shape (e.g., rectangular) oran arcuate shape such as circular or oval. For certain embodiments, thesubstrate is a glove such as described in U.S. Published Application No2006/0141014 which is incorporated herein in its entirety. In oneembodiment of the invention, the product includes a plurality ofwater-insoluble substrates of different shapes.

The present invention further comprises a method of improving thebarrier function and moisturization of skin by applying to skin in needof improving skin barrier function and moisturization an extract ofPichia anomala and n-acetyl glucosamine. The method comprises forexample topically applying a composition of the present inventioncomprising combined Pichia anomala extract and n-acetyl glucosamine toskin in need of improving skin barrier function and moisturization. Suchtopical application may be to any skin in need of treatment on the body,for example skin of the face, lips, neck, chest, back, arms, axilla,hands, feet and/or legs. The combined Pichia anomala extract andn-acetyl glucosamine are preferably applied in an effective amount thatresults in the desired improvement of skin barrier function beingachieved.

The present invention further comprises a method of improving theappearance of at least one sign of skin aging by applying to skin inneed of improving the appearance of at least one sign of skin agingcombined Pichia anomala extract and n-acetyl glucosamine. The methodcomprises for example topically applying a composition of the presentinvention comprising combined Pichia anomala extract and n-acetylglucosamine to skin in need of treatment of at least one sign of skinaging. Such topical application may be to any skin in need of treatmenton the body, for example skin of the face, lips, neck, chest, back,arms, axilla, hands, feet and/or legs. The combined Pichia anomalaextract and n-acetyl glucosamine are preferably applied in an effectiveamount that results in the desired improvement in the appearance of atleast one sign of skin aging being achieved.

Any suitable method of applying the composition to the skin in need maybe used. For example, the composition may be applied directly from apackage to the skin in need, by hand to the skin in need, or may betransferred from a substrate such as a wipe or mask, or a combination oftwo or more thereof. In other embodiments, the composition may beapplied via a dropper, tube, roller, spray, and patch or added to a bathor otherwise to water to be applied to the skin, and the like. Thecomposition may be applied in a variety of manners/forms, including,without limitation, as a leave-on cream, mask, and/or serum.

In certain embodiments, the methods of the present invention compriseapplying at least two different compositions or products comprisingPichia anomala extract and n-acetyl glucosamine to the skin. Forexample, the methods may comprise applying a first compositioncomprising an extract of Pichia anomala, followed by applying a secondcomposition comprising n-acetyl glucosamine that is different from thefirst composition, to the skin in need of treatment.

In certain preferred embodiments, the first and second composition maybe independently selected from the group consisting of lotions,cleansers, masks, wipes, creams, serums, gels, and the like. In certainpreferred embodiments, at least one of the first and second compositionsis a cleanser, lotion, cream, essence, or serum and the other is afacial mask or wipe. In certain other preferred embodiments, at leastone of the first and second compositions is a cleanser and the other isa lotion or cream.

The composition and formulations and products containing suchcompositions of the present invention may be prepared using methodologythat is well known by an artisan of ordinary skill. These compositionsmay be useful in treating skins of aging such as wrinkles, loss ofelasticity, uneven skin including reducing blotchiness. The compositionmay be used on a routine basis and is substantially free of skinirritants.

In one embodiment, the invention provides a method of increasing theamount of hyaluronic acid produced by skin when contacted with anextract of Pichia anomala, comprising contacting the skin with acomposition comprising the extract of Pichia anomala and n-acetylglucosamine. In this manner, a low level of extract of Pichia anomala,for example less than about 0.1, or less than about 0.08, for exampleabout 0.052, weight percent, may be used and still achieve good HAproduction. N-acetyl glucosamine may act as an activity booster for theextract of Pichia anomala in inducing HA production by skin.

The following non-limiting examples further illustrate the presentinvention.

Example 1

Hyaluronic acid (HA) production by human skin explants was determinedafter treating them with test Compositions 1-4 containing differentamounts of Pichia anomala extract as follows.

First, a gel was made containing the ingredients set forth in Table 1.

TABLE 1 INCI Weight % Phenoxyethanol; Methylparaben; 0.7 Ethylparaben;Propylparaben Carbomer 1 20% Sodium Hydroxide Solution QS (pH = 6) Water98.3

Compositions 1-4 were made by combining 2 wt % of the gel with differentamounts of a 5% aqueous solution of Pichia anomala extract, along withadditional ingredients. The Pichia anomala had been grown on kiwi plant.The 5% aqueous solution was treated as a 100% stock solution.

Compositions 1-4 contained the ingredients shown in Table 2.

TABLE 2 Composition 1 Composition 2 Composition 3 Composition 4 INCI(weight %) (weight %) (weight %) (weight %) Gel 2 2 2 2 CetearylAlcohol; 7 7 7 7 Cetearyl Glucoside Isononyl 8 8 8 8 IsononanoatePhenoxyethanol; 0.7 0.7 0.7 0.7 Methylparaben; Ethylparaben;Propylparaben 5% Aq. Solution of 2.5 (0.065 wt % 5 (0.13 wt % 7.5 (0.195wt % 10 (0.26 wt % Pichia anomala Pichia anomala Pichia anomala Pichiaanomala Pichia anomala extract extract) extract) extract) extract) Water79.8 77.3 74.8 72.3

HA production by Compositions 1-4 was determined using immunohistologyon normal explants of human skin from three donors (29, 30, 55 yearsold). Eight mm diameter punches were cut from the explants and depositedon pieces of sterile gauze and placed, one explant per well, in six wellplates with 3 mL of culture media. The culture media was sold under thetradename GIBCO DMEM/F-12 (ThermoFisher Scientific, Waltham, Mass.,catalog #11514436) with 1% GIBCO Penicillin-Streptomycin (ThermoFisherScientific, catalog #11528876) and 0.1% amphotericin B sold under thetradename FUNGIZONE (ThermoFisher Scientific, catalog #11510496).

For each test composition, 5 μl of test composition was applied to anexplant once a day for 5 days. An untreated explant sample was used asthe control. On day 7 the explants were recovered, wiped with a sterilegauze, then cut in half vertically and fixed in 4% paraformaldehyde(V/V). On day 8 the explants were dehydrated and embedded in paraffin.Each test composition was tested in triplicate.

The paraffinized slides were stripped with xylene and epitope retrievalwas carried out with PT link (Agilent, Santa Clara, Calif.) and targetretrieval solution sold under the tradename ENVISION Flex, High pH(Dako, DM828, Agilent, Santa Clara, Calif.). Slides were then rinsedwith wash buffer sold under the tradename ENVISION (Dako, DM831,Agilent, Santa Clara, Calif.) one time for 10 mins. Permeabilization andsaturation were done with PBS 0.3% Triton/5% goat serum (Dako, SantaClara, Calif., catalog #CP3418/X090710-8) for 30 mins, followed bylabeling with Hyaluronic Acid Binding Protein (“HABP” from Calbiochem,catalog #385911, Millipore Sigma, St. Louis, N.J.) overnight at 4° C.The next day the slides were rinsed with PBS three times for 5 minuteseach. Antibody was revealed with biotin-binding protein covalentlyattached to a fluorescent label sold under the tradename ALEXA FLUOR 488streptavidin (Invitrogen™ catalog #S11223) and staining of nuclei wasdone with DAPI solution (Dako, Santa Clara, Calif.) at 5 μg/ml for 30min at ambient temperature. Slides were then rinsed with PBS and mountedwith Fluoprep mounting medium (bioMerieux UK Ltd., UK catalog #75521).

Pictures of the skin sections were taken with an Olympus IX70Fluorescence microscope (Olympus Corporation, Japan) coupled to an imageanalysis system (NIS-Elements AR software, Nikon Instruments, Melville,N.Y.). Quantitative analysis of images was conducted with ImageJsoftware.

The results are shown in Table 3. The results are expressed as averagefluorescence intensity of the dermis (in Arbitrary Units (AU)).Fluorescence intensity (green) is proportional to the synthesis ofhvaluronic acid.

TABLE 3 Weight % of 5% Weight Delta of HAPB as Solution of Pichia % ofPichia compared to anomala extract anomala extract untreated controlComposition 1 2.5 0.065 10.37 Composition 2 5 0.13 17.73 Composition 37.5 0.195 15.24 Composition 4 10 0.26 15.41

These results suggest that maximum HA production by skin is obtainedusing about 0.13 wt % of the Pichia anomala extract. Above this amount,production of HA leveled off.

Example 2

Hyaluronic acid (HA) production by human skin explants was determinedafter treating them with Compositions 5-7 according to the inventioncontaining Pichia anomala extract and n-acetyl glucosamine.

Compositions 5-7 contained the ingredients shown in Table 4.

TABLE 4 Composition 5 Composition 6 Composition 7 Phase INCI (Weight %)(Weight %) (Weight %) A Water 69.4 69.4 69.4 A HydroxethylAcrylate/Sodium 1.2 1.2 1.2 Acryloyldimethyl Taurate Copolymer BSclerotium Gum 0.4 0.4 0.4 B Glycerin 4.0 4.0 4.0 C Cetearyl Olivate,Sorbitan olivate 1.5 1.5 1.5 C Chlorphenesin 0.2 0.2 0.2 D Water 15 9 9D N-acetylglucosamine 3.0 1.0 2.0 D Glycerin 1 1 1 D Glyceryl dilaurate0.5 0.5 0.5 D Steareth-10 0.5 0.5 0.5 E Dimethicone; Dimethicone 2.5 2.52.5 Crosspolymer E Dimethicone 3.0 3.0 3.0 E Dimethicone; Dimethiconol 22 2 F 5% Extract of Pichia anomala 1 (0.026 wt % 3 (0.078 wt % 2 (0.052wt % solution Pichia anomala Pichia anomala Pichia anomala extract)extract) extract) F Ethylhexylglycerin; 0.8 0.8 0.8 Phenoxyethanol G 20%Citric acid Solution QS pH = 5.5-6 QS pH = 5.5-6 QS pH = 5.5-6 G 20%Sodium Hydroxide Solution QS pH = 5.5-6 QS pH = 5.5-6 QS pH = 5.5-6

The compositions were prepared in phases. Phase A was prepared bycombining water and Hydroxethyl Acrylate/Sodium Acryloyldimethyl TaurateCopolymer in a kettle bell mixer with a propeller stirring and mixeduntil fully dispersed, smooth and uniform. Phase B was prepared bypre-mixing sclerotium gum and glycerin until uniform, then added to themain kettle and again mixed until smooth and uniform. The main kettlewas then heated to 70-75° C. Next, for Phase C, using a Greerco highspeed homogenizer the Chlorphensin, Cetearyl Olivate, and Sorbitanolivate were added to the main kettle and mixed until a smooth, uniformbulk was formed. The main kettle was then cooled down to 60° C. withsweep blade mixing. Phase D was prepared by combining the water andn-acetyl glucosamine in a separate kettle while heating to 65° C., thenthe glycerin, glycerin dilaurate, and steareth-10 were added, mixeduntil uniform and held at 65° C. Once Phase D was uniform, it was addedto the main kettle at 65° C. and mixed until uniform with sweep mixing.The mixture was then cooled to 40° C. In a second side kettle, Phase Ewas prepared by combining Dimethicone/Dimethicone Crosspolymer andDimethicone, mixed until uniform, and then added to the main kettle at40° C., followed by the addition of Dimethicone/Dimethiconol. In PhaseF, the extract of Pichia anomala solution, ethylhexylglycerin, andphenoxyethanol were added to the main kettle at 40° C. or less and mixeduntil uniform. Lastly, the main kettle was cooled to 30° C. The pH wasadjusted to 5.5-6 using 20% citric acid and/or 20% sodium hydroxide ifneeded (Phase G).

The compositions were evaluated according to the skin explant modeldescribed in Example 1 for change in HAPB production against untreatedcontrol. The results are shown in Table 5.

TABLE 5 Weight % N- Delta of HAPB as Weight % of Pichia Acetyl comparedto anomala extract Glucosamine untreated control Composition 5---0.026--- 3 14.35 Composition 6 --0.078--- 1 12.34 Composition 7---0.052-- 2 16.15

Although each of Compositions 5-7 contained 0.078 wt % or less of Pichiaanomala extract, they all stimulated HA production in the explants athigh levels in the presence of n-acetyl glucosamine. Composition 7provided particularly good results. Composition 7 contained only 0.052wt % Pichia anomala extract with 2 wt % n-acetyl glucosamine butprovided a delta of HABP over untreated of 16.15, which is unexpectedlygreater than the delta of HABP over untreated provided by Composition 1in Example 1 (10.37) containing about the same amount of Pichia anomalaextract (0.065 wt %).

We claim:
 1. A topical composition comprising (i) less than about 0.1weight percent of an extract of Pichia anomala and (ii) n-acetylglucosamine.
 2. The topical composition of claim 1, wherein the extractof Pichia anomala is prepared from a strain of Pichia anomala present onkiwi fruit or leaves.
 3. The topical composition of claim 1, wherein theextract of Pichia anomala is prepared from a strain of Pichia anomalapresent on sugar cane.
 4. The topical composition of claim 1, whereinthe weight ratio of the extract of Pichia anomala to n-acetylglucosamine in the topical composition is about 0.026.
 5. The topicalcomposition of claim 1 further comprising at least 4 weight percentglycerin.
 6. The topical composition of claim 1 having a pH of about 6.5or less.
 7. A topical composition comprising about 0.052 weight percentof an extract of Pichia anomala and about 2 weight percent of n-acetylglucosamine.
 8. The topical composition of claim 7 further comprisingabout 1 to about 2 weight percent of a mixture of cetearyl olivate andsorbitan olivate, about 4 to about 5 weight percent glycerin, and atleast about 65 weight percent water, and having a pH of less than about6.
 9. A method of treating a sign of skin aging, comprising topicallyapplying to skin in need of treatment for skin aging a topicalcomposition comprising (i) less than about 0.1 weight percent of anextract of Pichia anomala and (ii) n-acetyl glucosamine.
 10. A method ofimproving skin barrier function and moisturization, comprising topicallyapplying to skin in need of improving skin barrier function andmoisturization a topical composition comprising (i) less than about 0.1weight percent of an extract of Pichia anomala and (ii) n-acetylglucosamine.
 11. A method of increasing the amount of hyaluronic acidproduced by skin when contacted with an extract of Pichia anomala,comprising contacting the skin with a composition comprising (i) lessthan about 0.1 weight percent of an extract of Pichia anomala and (ii)n-acetyl glucosamine.
 12. The method of claim 11, wherein thecomposition contains about 0.052 weight percent of the extract of Pichiaanomala and about 2 weight percent of n-acetyl glucosamine.
 13. Themethod 12, wherein the composition further comprises about 1 to about 2weight percent of a mixture of cetearyl olivate and sorbitan olivate,about 4 to about 5 weight percent glycerin, and at least about 65 weightpercent water, and has a pH of less than about 6.